Due to their hydrophobic nature retinoids are associated with proteins in vivo. Such proteins are engaged in the transport of the vitamin from its storage site in the liver to vitamin A-requiring peripheral tissues. Moreover, the uptake by cells of the protein- associated vitamin and the intracellular transport of the retinoids are facilitated by proteins. The current proposal is aimed at elucidating the discrete steps involved in the transport of vitamin A to its ultimate site(s) of action. Vitamin A is mobilized from the liver by the retinol-binding protein (RBP). Attempts will be made to develop an in vitro system that allows detailed examination of how retinol is transferred across the membrane of the endoplasmic reticulum and is acquired by the nascent RBP. Moreover, stably transfected cell lines manufacturing RBP, grown in normal and vitamin A- deficient culture media, will be employed to unravel why retinol- deficient RBP molecules do not exit the endoplasmic reticulum. Based on the known three-dimensional structures of RBP and prealbumin, attempts will be made to map the sites of interaction between the two proteins using, in succession, antipeptide antibodies, site-directed mutagenesis and computer graphics modeling. The RBP-site interacting with a cell surface receptor will be identified by a similar approach. Most cells contain two intracellular-retinoid-binding proteins, CRBP and CRABP. Attempts will be made to obtain cDNA clones for CRABP and elucidate the structure of the CRABP gene. The possible role of CRBP in the intercellular transfer of vitamin A between fat storing cells of the liver and hepatocytes will be approached. CRBP-expression in isolated fat storing cells will be examined and the distribution of CRBP and RBP mRNAs in normal and vitamin A-deficient rat tissues will be studied using in situ hybridization techniques. The possibility the CRBP and CRABP deliver their ligands to as yet undiscovered intracellular structures will be examined by exploring the use of anti-idiotypic antibodies. This approach will be complimented by attempts to devise in vitro systems that allow transfer of retinoids from the intracellular binding proteins to subcellular structures.